Significance: The proposed research seeks to fill an important technological gap in anti-HIV drug discovery by developing a sensitive fluorescence polarization assay that can be automated for high throughput screening (HTS) for small molecule inhibitors of dimerization of the C-terminal domain of the HIV-1 capsid protein or CCA. Since CCA dimerization is critically important for self-association of the HIV Gag polyprotein and capsid formation, such inhibitors are expected to abrogate HIV assembly and maturation, promising a novel class of therapeutic agents for the treatment of HIV-1 infection. Background: FDA-approved existing anti-HIV drugs span six mechanistic classes and target reverse transcriptase, protease, integrase, fusion, and entry. However, resistance to all these drug classes has been documented in vitro and in vivo. New therapies that target different viral components are urgently needed to combat expanding global HIV epidemics. HIV assembly and maturation, while a significant antiviral target, has remained largely unexploited. To facilitate antiviral drug discovery that targets HIV assembly and maturation, accurate, sensitive and reproducible assays suitable for HTS of compound libraries have yet to be developed. Objective/Hypothesis: We seek to develop a fluorescence polarization assay suitable for HTS for small molecule inhibitors of HIV assembly and maturation. Specific Aims: (1) Establish and optimize a fluorescence polarization assay for HTS for small molecule inhibitors of HIV capsid assembly; (2) Screen FDA-approved drug libraries, and biochemically as well as structurally validate the positive hits as authentic inhibitors of CCA dimerization; (3) Test the hypothesis that CCA dimerization inhibitors suppress HIV-1 replication in primary target cells. Study design: We will site-specifically label CCA with a suitable fluorescent dye, and use the fluorescent protein as a probe for quantifying the CCA dimer-to-monomer transition in the presence of library compounds using fluorescence polarization techniques. We will conduct primary HTS of FDA-approved drug libraries in a 384-well format, and structurally and functionally validate the positive hits before testing their ability to inhibit assembly of both mature-like and immature-like particles in vitro. Active compounds will be tested for their ability to inhibit HIV replication in primary target cells. Our long-term objective is to develop HIV assembly and maturation inhibitors as a novel class of therapeutic agents for the prevention of HIV-1 infection and the treatment of AIDS. PUBLIC HEALTH RELEVANCE: Current antiretroviral therapy (ART) for HIV-1 infected patients utilizes a combination of inhibitors that target the viral enzymes reverse transcriptase and protease. ART reduces viral load and slows the progression of HIV to AIDS, contributing to a steady decrease in AIDS deaths in the world. Despite its success, ART does not eradicate HIV from infected cells, and, among many complications of ART is the emergence of drug-resistant HIV strains not responding to current antiretroviral regimens. The proposed research aims to develop a sensitive fluorescence polarization assay that can be automated for high-throughput screening for small molecule inhibitors of dimerization of the C- terminal domain of the HIV-1 capsid protein. Such inhibitors may become additional weapons in the arsenal to fight HIV infection by specifically targeting HIV assembly and maturation.